In 2PE, two photons of lower energy (typically in the infrared range) are absorbed simultaneously by a fluorophore. This combined energy is sufficient to excite the fluorophore, which then emits light at a shorter wavelength (usually in the visible range). The key to 2PE is that the probability of two-photon absorption is highest at the focal point of the laser, allowing for pinpoint excitation and reducing out-of-focus light. This leads to enhanced image clarity and depth.
